The long-term objectives of this research project are to help define the molecular basis of meningococcal endotoxin (lipooligosaccharide [LOS]), a critical virulence factor of the human pathogen Neisseria meningitidis. Meningococcal infections remain devastating in the United States and worldwide. Current meningococcal vaccines have major limitations and antibiotic-resistant meningococci are emerging, emphasizing the need to better understand the molecular mechanisms involved in meningococcal pathogenesis. This application is directed at understanding the structural-functional relationships and heterogeneity of the meningococcal LOS inner core, Hep/2[GlcNAc/(0/1), Glc/(0/1)] PEA/(0/1/2)-KDO/2 Lipid A. LOS inner core structure influences LOS assembly, human immune recognition of the meningococcus and the interactions of LOS with soluble and cellular human proteins. The goals are to understand meningococcal LOS inner core biosynthesis and how meningococcal LOS inner core structure activates TLR4 and influences recognition by human antibody. In Specific Aim 1, the molecular basis of the meningococcal LOS inner core assembly and heterogeneity will be further characterized by: (a) structural and genetic analysis of defined mutants that express only lipid A; (b) identification and characterization of meningococcal genes necessary for the variable phosphorylation of meningococcal lipid A; (c) identification and characterization of meningococcal genes involved in the addition and/or removal of phosphate of Hep II. In Specific Aim 2, the importance of meningococcal LOS inner core structure and heterogeneity in: (a) activation of human Toll 4 receptor (TLR4), and (b) recognition of meningococci by human antibodies will be determined. To accomplish these Aims, genetically defined LOS mutants of meningococci; analytical biochemistry and purified LOS structures; and established human cell and tissue assays will be utilized. Data obtained from the proposed studies has application to the design of meningococcal vaccines currently in development (e.g., inclusion of truncated LOS molecules in outer membrane vesicle or conjugate vaccines) and to the development of therapeutic agents that may treat meningococcal sepsis and meningitis. The studies will also continue to define differences between enteric endotoxin and the unique structure and biology of meningococcal endotoxin.